The invention relates to the area of bacterial genetic engineering. In particular, it relates to the bacteria Pasteurella haemolytica. 
The microorganism P. haemolytica biotype A, serotype 1, is the principal causative agent of pneumonic pasteurellosis in cattle. If techniques could be developed for introducing exogenous DNA into P. haemolytica, it would be possible to produce site-specific mutations in this bacterium. Such mutants could provide xe2x80x9crationallyxe2x80x9d attenuated strains for use as live vaccines.
Attenuated auxotrophic mutants were first described by Bacon and Burrows in the early 1950""s. They reported that attenuated auxotrophs of Salmonella typhi defective in the aromatic amino acid biosynthetic pathway were avirutent in mice. Subsequently, it has been demonstrated in widely diverse bacteria that disrupting the aromatic amino acid biosynthetic pathway produces attenuated organisms. For example, attenuated strains of the invasive bacteria Salmonella typhi, Salmonella ryphimurium, Shigella flexneri, and Yersina enterocolitica, were generated by introducing mutations in their respective aroA genes. Also attenuation was produced in the non-invasive bacteria Bordetella pertussis and Pasteurella multocida through aroA inactivation. Strains which carry aroA mutations are unable to synthesize chorismic acid from which p-aminobenzoic acid, dihydrobenzoate, and aromatic amino acids are produced. It is likely that the absence of one or more of these compounds in vivo is responsible for the poor growth of aroA mutants in the hosts.
Live attenuated bacterial strains generally provide superior protection as compared to killed bacterial vaccines (bacterins). In general, live vaccines elicit a stronger cell mediated response in the host than do bacterins. The superior immunity provided by attenuated live organisms may be explained by their ability to induce expression of stress-proteins and, possibly, of certain toxins within the host. The immune response generated by live organisms would be directed against these abundant proteins and thereby provide better protection.
There is a long-felt and continuing need in the art for veterinary vaccines to protect cattle from P. haemolytica infection. There also is a need for techniques for introducing DNA into P. haemolytica. 
It is an object of the invention to provide methods for mutagenizing P. haemolytica. 
It is another object of the invention to provide a P. haemolytica gene for production of an enzyme for use in preparing genetic material for introduction into P. haemolytica. 
It is yet another object of the invention to provide an enzyme for use in preparing genetic material for introduction into P. haemolytica. 
It is still another object of the invention to provide a plasmid for unstable introduction of genetic material into P. haemotytica. 
It is an object of the invention to provide P. haemoltica mutant strains.
It is another object of the invention to provide live, attenuated vaccines against P. haemotyrica infection.
It is another object of the invention to provide genetically engineered P. haemolytica. 
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention a method for site-directed mutagenesis of P. haemolytica is provided. The method comprises the steps of: isolating a DNA region from P. haemolytica in which region a mutation is desired; introducing a mutation into said DNA region to form a mutated DNA region; methylating said mutated DNA region with a methylating enzyme, to form methylated DNA, which methylated DNA is refractory to endonuclease cleavage at GATGC and GCATC sequences; introducing said methylated DNA into P. haemolytica to form transformants; and screening said transformants for those which have said mutation in said region on chromosomal DNA of said P. haemolytica cell.
In an alternative embodiment of the invention site-directed mutagenesis of P. haemolytica is accomplished by the steps of: isolating a DNA region from P. haemolytica in which region a mutation is desired; introducing a mutation into said DNA region to form a mutated DNA region; introducing said methylated DNA into a P. haemolytica cell which does not express a PhaI restriction endonuclease, to form transformants; and screening said transformants for those which have said mutation in said region on chromosomal DNA of said P. haemolytica cell.
In another embodiment of the invention an isolated and purified gene is provided. The gene encodes PhaI methyltransferase.
In still another embodiment of the invention another isolated and purified gene is provided. The gene encodes PhaI restriction endonuclease.
In yet another embodiment of the invention a preparation of PhaI methyltransferase is provided. The preparation is free from PhaI restriction endonuclease.
In still another embodiment of the invention a preparation of PhaI restriction endonuclea is provided. The preparation is free from PhaI methyltransferase.
In another embodiment of the invention a chimeric plasmid is provided which is suitable for unstable introduction of genetic material into P. haemolytica. The plasmid comprise a 4.2 kb P. haemolytica plasmid encoding a streptomycin resistance determinant deposited at the American Type Culture Collection as Accession No. ATCC 69499; and a plasmid which cannot replicate in P. haemolytica. 
In an additional embodiment of the invention a P. haemolytica mutant is provided. The mutant is made by the process of the invention described in more detail below.
In another embodiment of the invention a P. haemolytica mutant is provided which does not express the PhaI restriction endonuclease.
In another embodiment of the invention a P. haemolytica aroA mutant is provided.
In still another embodiment of the invention a vaccine is provided. The vaccine comprises an attenuated, live, mutant of P. haemolytica which has an aroA mutation.
In yet another embodiment of the invention an isolated and purified P. haemolytica strain is provided. The strain has been genetically modified by the introduction of DNA.
These and other embodiments of the invention provide the art with the means to construct desirable mutants of the economically important and previously intractable pathogen P. haemotytica.